The classic paradigm for understanding the immunobiology of cutaneous vaccination is that antigen delivered with an adjuvant creates an inflammatory stimulus, which in turn induces epidermal Langerhans cells to mature and to migrate to draining lymph nodes. However, repeat vaccination with melanoma peptides in adjuvant is associated with dramatic chronic local inflammation and may have different biology. Chronic inflammation in autoimmune and infectious diseases can lead to new lymphoid tissues in locations that are not classic lymphoid areas. This process, termed lymphoid neogenesis, can result in tertiary lymphoid organs (TLO) that function like lymph nodes. We have observed lymph node-like aggregates (LNLA) in the dermis of human skin after a single injection of an emulsion of incomplete Freund's adjuvant plus GM-CSF. These LNLA contain mature CD83+DC-LAMP+ dendritic cells, T cells, B cells, and peripheral node addressin (PNAd) expressing microvessels suggestive of high endothelial venules. Preliminary studies in sites of repeated peptide vaccination with adjuvant show other features of lymphoid neogenesis, but there also is evidence of regulatory cells in the aggregates. We hypothesize that: (i) repeat vaccination with melanoma peptides and adjuvant may cause persistence of LNLA and creation of tertiary lymphoid organs at vaccine sites, (ii) extending immunization may result in a secondary set of immune regulatory influences, which could be blocked in a targeted manner if their prevalence and timing is defined, and (iii) antigen specific CD4+ and CD8+ T cells are induced locally at vaccine sites and may be generated by direct introduction of peptide antigens into a pre-induced immunization microenvironment. Thus, for the current proposal, we propose to perform 2 clinical trials combining immunization with a well- characterized multipeptide vaccine with extensive studies of tissue obtained from a replicate immunization site. Specific aims are: Aim 1. To determine whether repeat immunization (1-3 times) with Adjuvant with or without peptides induces lymph-node like aggregates (LNLA) and tertiary lymphoid organs (TLO) in skin, Aim 2. To determine whether extended immunization (4-6 vaccines) is associated with induction of immune regulatory processes in the vaccination site microenvironment /tertiary lymphoid organs, Aim 3. To characterize antigen-reactive CD4 and CD8 T cells in sites of immunization with a multipeptide vaccine. The results of this work will likely change our understanding of the immunobiology in the immunization microenvironment and identify approaches for combination therapies to improve antitumor immunity. PUBLIC HEALTH RELEVANCE: Cancer vaccines hold promise as a low toxicity treatment that may harness the immune system's response to cancer, and to melanoma in particular. Melanoma vaccines can induce immune responses in many or most patients, but the clinical impact has been disappointing. The present proposal is to characterize cellular and molecular events associated with induction of immune responses by melanoma vaccines, with the intent of improving the therapeutic potential of vaccines through a better understanding of these events.